Reagent for measuring alanine aminotransferase activity

ABSTRACT

A reagent for measuring an alanine aminotransferase activity comprising L-alanine, 2-oxoglutaric acid, lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide, characterized by further comprising a substance having an activity of inhibiting lactate dehydrogenase activity is disclosed. Further, a method for measuring an alanine aminotransferase activity, characterized by bringing a sample to be analyzed, which may contain alanine aminotransferase, into contact with L-alanine, 2-oxoglutaric acid, lactate dehydrogenase, reduced nicotinamide adenine dinucleotide, and a substance having an activity of inhibiting a lactate dehydrogenase activity is disclosed. According to the reagent and method, an increase in the reagent blank reaction, i.e., an increase in the initial absorbance, can be suppressed.

This application is a 371 of PCT/JP03/02897 filed Mar. 12,2003.

TECHNICAL FIELD

The present invention relates to a reagent for measuring an alanineaminotransferase activity.

BACKGROUND ART

Alanine aminotransferase (hereinafter referred to as ALT) is an enzymeabundantly distributed in the heart or liver. Because ALT is released toblood during a disease, the measurement of an ALT activity in a bodyfluid such as urine or blood is important as a marker for a diagnosis ofa heart or liver disease or observation after treatment thereof.

In a method commonly used for measuring the ALT activity, pyruvic acid,which is generated from L-alanine and 2-oxoglutaric acid as substratesby ALT, is changed to lactic acid by lactate dehydrogenase (hereinafterreferred to as LD), and a decreased amount of reduced nicotinamideadenine dinucleotide (hereinafter referred to as NADH) coexisting ismeasured at the wavelength of approximately 340 nm.

The reaction formulae are as follows:

In the above formula, NAD means oxidized nicotinamide adeninedinucleotide.

As previously described, the measurement of ALT activity is important asa marker for a diagnosis of a heart or liver disease or observationafter treatment thereof, and is carried out internationally. However,because various methods for measuring the ALT activity are known andused, different values obtained in accordance with different facilitiesor persons lack compatibility, and thus there is concern that problemsin clinical diagnosis may arise. Therefore, recommendations in whichreaction principles, the composition of reagents, concentrations ofreagents, and the like are prescribed are suggested in each country, andthe ALT activity is thus measured in each facility, to ensurecompatibility with values measured in accordance with therecommendations.

In Japan, the Japan Society of Clinical Chemistry (JSCC) published in1989 a recommendation for measuring the ALT activity [JSCC:Recommendation for measuring enzyme activities in human serum-alanineaminotransferase-(1989-08-30), Japanese Journal of Clinical Chemistry,18(4), 250–262 (1989)].

The recommendation published by JSCC is a method in which common enzymeactivities are measured under optimal conditions, which may be shared bytechnical levels in a clinical laboratory or the like, to ensure thecompatibility of measuring values, and thus it cannot be generally usedas a daily test. Therefore, manufacturers of reagents for clinicallaboratory test supply reagents capable of ensuring compatibilitybetween measuring values and the JSCC recommendation, and have conductedintensive studies into the provision of stable and low-cost reagents toobtain accurate and precise measuring values and the compatibilitybetween measuring values and the JSCC recommendation.

In facilities such as a clinical laboratory where the ALT activity ismeasured, reagents for measuring the ALT activity, which are supplied bythe manufacturers of reagents for clinical laboratory test and ensurethe compatibility between measuring values and the JSCC recommendation,are generally used as a daily test. However, because samples from aliving body are mixtures of various components and reagents formeasuring the ALT activity are also mixtures of various components, itis very difficult to develop a reagent which is stable and not affectedby impurities, for measuring the ALT activity.

Particularly, in an automatic analyzer in which only the setting ofmeasuring reagents and the setting of measuring conditions are needed,measurement is often carried out after keeping reagents open for severalweeks. In this case, the reagents for measuring the ALT activity absorbcarbon dioxide in air, and then pH in the reagents followed by thereagent blank reaction changes, to produce a problem, i.e., errors inthe measuring values of ALT activity obtained.

Therefore, the object of the present invention is to provide a reagentfor measuring the ALT activity which can suppress an increase in thereagent blank reaction, i.e., an increase in the initial absorbance.

DISCLOSURE OF THE INVENTION

The object can be solved by the present invention, i.e., a reagent formeasuring an alanine aminotransferase activity comprising L-alanine,2-oxoglutaric acid, lactate dehydrogenase, and reduced nicotinamideadenine dinucleotide, characterized by further comprising a substancehaving an activity of inhibiting a lactate dehydrogenase activity.

According to a preferred embodiment of the present invention, thereagent for measuring an alanine aminotransferase activity is a tworeagent-components system, and contains the substance having an activityof inhibiting a lactate dehydrogenase activity in either of a firstreagent-component or a second reagent-component or both of the first andsecond reagent-components.

According to another preferred embodiment of the present invention, thereagent for measuring an alanine aminotransferase activity is a tworeagent-components system, and contains lactate dehydrogenase and thesubstance having an activity of inhibiting a lactate dehydrogenaseactivity in the same reagent-component.

According to still another preferred embodiment of the presentinvention, the reagent for measuring an alanine aminotransferaseactivity is a two reagent-components system, and contains at leastlactate dehydrogenase in a first reagent-component and at least2-oxoglutaric acid in a second reagent-component.

According to still another preferred embodiment of the presentinvention, the substance having an activity of inhibiting a lactatedehydrogenase activity is oxamic acid or a salt thereof.

Further, the present invention relates to a method for measuring analanine aminotransferase activity, characterized by bringing a sample tobe analyzed, which may contain alanine aminotransferase, into contactwith L-alanine, 2-oxoglutaric acid, lactate dehydrogenase, reducednicotinamide adenine dinucleotide, and a substance having an activity ofinhibiting a lactate dehydrogenase activity.

Furthermore, the present invention relates to a method for measuring analanine aminotransferase activity, characterized by comprising the stepsof:

bringing a sample to be analyzed, which may contain alanineaminotransferase, into contact with L-alanine, 2-oxoglutaric acid,lactate dehydrogenase, reduced nicotinamide adenine dinucleotide, and asubstance having an activity of inhibiting a lactate dehydrogenaseactivity, and measuring a decreased amount of reduced nicotinamideadenine dinucleotide or an increased amount of oxidized nicotinamideadenine dinucleotide generated.

According to a preferred embodiment of the measuring method of thepresent invention, the substance having an activity of inhibiting alactate dehydrogenase activity is oxamic acid or a salt thereof.

According to another preferred embodiment of the measuring method of thepresent invention, a concentration of oxamic acid or a salt thereof is0.005 to 5 mmol/L as a final concentration in a measuring system.

According to still another preferred embodiment of the measuring methodof the present invention, a concentration of lactate dehydrogenase is100 U/L or more as a final concentration in a measuring system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a graph showing a time course for a blank sensitivity in thereagents for measuring an ALT activity of the present invention and forcomparison.

FIG. 2 is a graph showing a time course for the ALT activity in a pooledserum measured using the reagents for measuring an ALT activity of thepresent invention and for comparison.

FIG. 3 is a graph showing an LD stability of the reagents for measuringan ALT activity of the present invention and for comparison.

BEST MODE FOR CARRYING OUT THE INVENTION

The reagent of the present invention for measuring an ALT activity is animproved reagent of a known reagent for measuring an ALT activitycomprising L-alanine, 2-oxoglutaric acid, LD, and NADH. In the reagentfor measuring an ALT activity comprising L-alanine, 2-oxoglutaric acid,LD, and NADH, pyruvic acid, which is generated from L-alanine and2-oxoglutaric acid as substrates by ALT, is changed to lactic acid byLD, and a decreased amount of NADH coexisting or an increased amount ofNAD generated is measured at the wavelength of approximately 340 nm.

The reagent for measuring an ALT activity of the present inventioncomprises a substance having an activity of inhibiting an LD activity(hereinafter referred to as an LD inhibitor) in addition to the knowncomponents. The LD inhibitor used in the present invention is notparticularly limited, but there may be mentioned, for example, oxamicacid, oxalic acid, oxalacetic acid, pyruvic acid, phosphoenolpyruvicacid, sodium dodecyl sulfate, lactic acid, or hydroxyglutaric acid, orsalts thereof. Oxamic acid or a salt thereof such as sodium salt,potassium salt, or lithium salt, usually does not lead to errors inmeasuring an ALT activity, and thus is preferable.

A concentration of the LD inhibitor contained in the reagent formeasuring an ALT activity of the present invention may be changed inaccordance with the kind of the LD inhibitor used, and thus is notparticularly limited, so long as it is the concentration exhibiting theLD activity which does not affect a reagent for measuring an ALTactivity. The final concentration in a measuring system may be generally0.001 to 100 mmol/L, obtained by adjusting the concentration containedin the measuring reagent.

The term “exhibiting the LD activity which does not affect a reagent formeasuring an ALT activity” as used herein means that at least an LDactivity capable of measuring the ALT activity remains in a sample to beanalyzed. Even if the LD activity is inhibited by the LD inhibitor, sucha reagent for measuring an ALT activity can be used, so long as aminimum LD activity enough for measurement remains. In addition, theobject of the present invention is to suppress the reagent blankreaction, and thus appropriate amounts of LD and the LD inhibitor can beselected, so as to exhibit the LD activity which does not affect areagent for measuring an ALT activity and to reduce the reagent blankreaction to as low as possible.

More particularly, for example, when oxamic acid is used as the LDinhibitor, the desired effect can be obtained by adding oxamic acid sothat the final concentration in the measuring system becomes preferably0.005 to 5 mmol/L, more preferably 0.02 to 1 mmol/L. As described below,when the reagent components are divided into the first reagent-componentand the second reagent-component, the same effect can be obtained byadding oxamic acid to either of the first reagent-component or thesecond reagent-component or both of the first and secondreagent-components, so long as the final concentration is within theabove range. Further, when the LD inhibitor coexists with LD, LD can bestabilized.

Among the components contained in the reagent for measuring an ALTactivity of the present invention, each component contained in knownreagents for measuring an ALT activity, i.e., LD, NADH, L-alanine, and2-oxoglutaric acid, can be used in a manner similar to that of knownreagents.

For example, a naturally occurring LD, such as that derived from chickenheart, pig heart, pig muscle, or Leuconostoc mesenteroides, or arecombinant LD thereof, can be used as LD. Therefore, the origin thereofis not particularly limited.

The concentration of LD contained in the reagent for measuring an ALTactivity of the present invention can be appropriately selected so thatthe final concentration in a measuring system becomes at least 100 U/Lor more. In this connection, the reagent for measuring an ALT activityof the present invention is characterized by further comprising the LDinhibitor, and thus the concentration can be appropriately selected sothat the final concentration of the LD activity becomes at least 100 U/Lor more in consideration of the LD inhibition by the LD inhibitor. Forexample, when oxamic acid as the LD inhibitor is added, so that theconcentration becomes 0.02 to 1 mmol/L, an appropriate amount of LD canbe used so that the final concentration of the LD activity becomes atleast 100 U/L or more.

The term “LD activity” as used herein means an activity of reducingpyruvic acid to lactic acid. The unit “U” is defined as the amount ofenzyme activity obtained when converting 1 μmol of the substrate(pyruvic acid) to the product (lactic acid) for a minute (standardtemperature 30° C.).

The concentration of NADH contained in the reagent for measuring an ALTactivity of the present invention may be preferably 0.05 to 2 mmol/L,more preferably 0.1 to 0.5 mmol/L, as the final concentration in ameasuring system.

The concentration of L-alanine as one of the substrates may bepreferably 100 to 3000 mmol/L, more preferably 200 to 2000 mmol/L, asthe final concentration in a measuring system.

The concentration of 2-oxoglutaric acid as another substrate may bepreferably 1 to 500 mmol/L, more preferably 5 to 100 mmol/L, as thefinal concentration in a measuring system.

The reagent for measuring an ALT activity of the present invention maycontain an appropriate buffer, as known reagents for measuring an ALTactivity of the present invention. The buffer used may be a buffer whichdoes not cause an adverse effect when measuring the ALT activity and canbe appropriately selected from known buffers. There may be mentioned,for example, tris(hydroxymethyl)aminomethane, phosphoric acid,2-[4-(2-hydroxyethyl)-1-piperazyl]ethanesulfonic acid,bis(2-hydroxymethyl)iminotris(hydroxyethyl)methane,2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid,N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, orn-ethylmorpholine.

The reagent for measuring an ALT activity of the present invention mayoptionally contain, in addition to the essential components and thebuffer, commonly used components such as chelating agents [for example,ethylenediaminetetraacetic acid (EDTA)], preservatives (for example,azide), stabilizers (for example, albumin or glycerol), and/ordetergents.

The combination of reagent components in the reagent for measuring anALT activity of the present invention is not particularly limited, asknown reagents for measuring an ALT activity, and may be a onereagent-component system or a two reagent-components system. Generally,a reagent in which the components are divided into stable conditions andreactions can be carried out under optimal conditions for measuring theactivity is preferred. In such a combination, for example, the firstreagent-component containing NADH, LD, and the like, which are stableunder alkaline conditions, and the second reagent-component containing2-oxoglutaric acid and the like are prepared, so that the concentrationsthereof and pH become optimum for measuring the activity duringreactions, and the LD inhibitor and 1-alanine can be added to either ofthe first reagent-component or the second reagent-component or both ofthe first and second reagent-components. However, the present inventionis not particularly limited to these combinations.

When measuring a sample to be analyzed, which may contain pyruvic acid,for example, a body fluid (such as blood, serum, plasma, or urine),cells, or tissues, a two reagent-components system in which at least LDis contained in the first reagent-component and 2-oxoglutaric acid iscontained in the second reagent-component is preferable, to removeeffects due to pyruvic acid derived from the sample. The ALT activitycan be measured without effects due to pyruvic acid derived from thesample, by mixing the sample which may contain pyruvic acid with thefirst reagent-component containing LD to eliminate pyruvic acid derivedfrom the sample, and adding the second reagent-component thereto.

In this connection, in the case of a two reagent-components system inwhich at least LD and 2-oxoglutaric acid are contained in the secondreagent-component, the ALT activity can be measured by adding the secondreagent-component and incubating for a member of seconds toapproximately a minute to eliminate pyruvic acid derived from thesample. Alternatively, in the case of a one reagent-component system,the ALT activity can be measured. Therefore, the reagent for measuringan ALT activity of the present invention is not limited to the specificcombinations of reagent components.

The reagent of the present invention for measuring an ALT activity canbe used, for example, in the method of the present invention formeasuring an ALT activity. In the method of the present invention formeasuring an ALT activity, the ALT activity can be measured by bringinga sample to be analyzed into contact with L-alanine, 2-oxoglutaric acid,LD, NADH, and an LD inhibitor, and measuring a decreased amount of NADHor an increased amount of NAD generated at the wavelength ofapproximately 340 nm.

The sample to be analyzed is not particularly limited, so long as it isa sample which may contain the ALT activity. There may be mentioned, forexample, body fluids commonly used in clinical diagnosis (such as blood,serum, plasma, or urine), cells, tissues, or experimental samples.

Mechanisms

The present inventor considers the reason for the suppressing of thereagent blank reaction in the reagent for measuring an ALT activity ofthe present invention, as follows. However, the present invention is notlimited to the following hypothesis.

It is known that LD contained in the reagent for measuring an ALTactivity of the present invention has not only a dehydrogenase activityto lactic acid as a major enzyme activity, but also an activity ofreducing 2-oxoglutaric acid, i.e., a 2-hydroxyglutaric aciddehydrogenase (HGD) activity [Japanese Journal of Clinical Chemistry,18(4), 250–262 (1989)]. The reaction is as follows:

As previously described in BACKGROUND ART, in widely used methods formeasuring the ALT activity, including the recommendation published bythe Japan Society of Clinical Chemistry, 2-oxoglutaric acid is used as asubstrate. Therefore, when LD has the dehydrogenase activity to2-oxoglutaric acid (i.e., HGD activity), NADH is oxidized, and thereagent blank and the reagent blank reaction are increased.

Further, the dehydrogenase activity to 2-oxoglutaric acid is changed bya pH change, to generate errors in the obtained measuring values of theALT activity. For example, in an automatic analyzer in which only thesetting of measuring reagents and the setting of measuring conditionsare needed, measurement is often carried out after keeping reagents openfor several weeks. In this case, the reagents for measuring the ALTactivity absorb carbon dioxide in air, and then pH in the reagentsfollowed by the reagent blank reaction changes, to generate errors inthe obtained measuring values of the ALT activity.

Furthermore, the dehydrogenase activity to 2-oxoglutaric acid produces aproblem in that an apparent Km of LD to pyruvic acid is increased duringa measuring of the ALT activity, to cause negative errors in themeasured values of the activity. It is considered that the reagent blankreaction is a reaction in which 2-oxoglutaric acid is reduced by LD perse, i.e., the HGD activity.

The present inventor confirmed that LD exhibits the HGD activity andthat the reagent blank is changed in accordance with an amount of LDadded in the reagent for measuring an ALT activity. Further, the optimumpH of the HGD activity is between a weak acidity and around neutral. Forexample, when an amount of LD is increased, the reagent blank isincreased. In addition, when measuring the ALT activity under weakalkaline conditions, the reagent blank is increased and accuratemeasured values of the ALT activity cannot be obtained, due to adecreased pH of reagents due to keeping the reagents open.

It is considered that the reagent blank can be reduced by decreasing anamount of LD. However, if the amount of LD is too small, an amount of LDrequired to measure the ALT activity is insufficient, and thus aquantitativeness cannot be obtained. Further, an increased reagent blankdue to a decreased pH of reagents caused by keeping reagents open can beavoided by changing the pH during the measuring of the ALT activity toalkaline conditions. However, this is not appropriate, because theoptimum pH of the ALT activity is a weak alkaline.

An inhibitor of the HGD activity has not been known until now, and underthese circumstances, the present inventor considered the following. Thatis, an LD inhibitor is added to a known reagent for measuring an ALTactivity, to inhibit the HGD activity of LD per se and, as a result, toreduce the reagent blank without the loss of quantitativeness. Further,an increased reagent blank due to a decreased pH of reagents caused bykeeping reagents open can be avoided, and negative errors in measuredvalues of the ALT activity due to an increased apparent Km of LD topyruvic acid can be reduced.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples.

Preparative Example 1

As the reagent for measuring an ALT activity of the present invention, atwo reagent-components system composed of the first reagent-componentcontaining the components shown in Table 1 and the secondreagent-component containing the components shown in Table 2 wasprepared.

Further, as a known reagent for measuring an ALT activity forcomparison, a two reagent-components system, which was the same as thatof the reagent of the present invention except that oxamic acid was notcontained in the first reagent-component, was prepared.

Hereinafter, the two reagent-components system for comparison isreferred to as “reagent A” and the two reagent-components system of thepresent invention is referred to as “reagent B”. The prepared reagents Aand B were transferred to sealed containers and kept therein until usedin the following Example for evaluation.

TABLE 1 Concentration Components 20 mmol/L Tris-HCl (pH 9.20) 200 mmol/LL-alanine 0.5 mmol/L oxamic acid 0.095% sodium azide 0.25 mol/L NADH 3KU/L LD (recombinant LD; Oriental Yeast Co., Ltd.)

TABLE 2 Concentration Components 360 mmol/L Tris-HCl (pH 4.50) 1080mmol/L L-alanine 63 mmol/L 2-oxoglutaric acid 0.01% EDTA2NaExample for Evaluation 1: Evaluation of Reagent for Measuring an ALTActivity of the Present Invention(1) Measurement of Reagent Blank

Reagent containers for an automatic analyzer (7170S; Hitachi Ltd.) werefilled with the first reagent-component (60 mL) and the secondreagent-component (20 mL) of the reagent A for comparison, and the firstreagent-component (60 mL) and the second reagent-component (20 mL) ofthe reagent B of the present invention prepared in Preparative Example1, respectively. The reagent containers were set in the automaticanalyzer and allowed to stand for 5 weeks. The automatic analyzer usedin the evaluation had a cooling unit and the reagent containers werekept at approximately 10° C. The reagent containers were kept open.

Immediately after the standing, and after 1 week, 2 weeks, 3 weeks, 4weeks, and 5 weeks, each reagent blank of the reagents A and B wasmeasured in accordance with the following procedure.

More particularly, to each reaction cell in the automatic analyzer, 7.5μL of physiological saline as a sample followed by 150 μL of the firstreagent-component were added, stirred, and incubated at 37° C. for 5minutes, and then 50 μL of the second reagent-component was furtheradded, stirred, and incubated at 37° C. for 5 minutes. Afterapproximately 1 minute to 5 minutes (i.e., for 4 minutes) from theaddition of the second reagent-component, an amount of change inabsorbance at the wavelength of 340 nm was measured, and an amount ofchange in absorbance per minute was calculated as a blank sensitivity.The results are shown in FIG. 1.

As shown in FIG. 1, during the keeping of the reagent containers open,the reagent blank in the reagent A for comparison was increased withtime, while that in the reagent B of the present invention showed littlechange.

(2) Measurement of ALT Activity in Pooled Serum

As similar to the above Example for evaluation 1(1), the reagents A andB were allowed to stand for 5 weeks. Immediately after the standing, andafter 1 week, 2 weeks, 3 weeks, 4 weeks, and 5 weeks, the ALT activityin a sample (a pooled serum) was measured in accordance with thefollowing procedure.

More particularly, 7.5 μL of a pooled serum or physiological saline(reagent blank) followed by 150 μL of the first reagent-component wereadded, stirred, and incubated at 37° C. for 5 minutes, and then 50 μL ofthe second reagent-component was further added, stirred, and incubatedat 37° C. for 5 minutes. After approximately 1 minute to 5 minutes(i.e., for 4 minutes) from the addition of the second reagent-component,an amount of change in absorbance at the wavelength of 340 nm wasmeasured, and an amount of change in absorbance per minute wascalculated. The ALT activity was calibrated on the basis of measuredvalues using an enzyme calibrator (INTERNATIONAL REAGENTS CORPORATION)instead of the pooled serum.

The results are shown in FIG. 2. As shown in FIG. 2, with respect tomeasured values of the pooled serum, the measured values of the ALTactivity in the reagent A for comparison were increased with time, whilethose in the reagent B of the present invention showed little change,during the keeping of the reagent containers open.

Example for Evaluation 2: Evaluation of LD Stability in Reagent forMeasuring an ALT Activity of the Present Invention

In the present example for evaluation, a time course for a residualratio of the LD activity in each first reagent-component was examined bythe following procedure. That is, 60 mL of the first reagent-componentof the reagent A for comparison and 60 mL of the first reagent-componentof the reagent B of the present invention prepared in PreparativeExample 1 were transferred to 70 mL-volume polyethylene bottles withcaps, respectively. After the closed bottles were incubated at 25° C. or37° C. for 3 weeks, a time course was examined.

More particularly, immediately after the incubation, and after 1 week, 2weeks, and 3 weeks, each reagent was collected. For measurement, eachreagent was diluted to 1/10 with 50 mmol/L phosphate buffer (pH 7.50)containing 0.1% bovine serum albumin as a sample, and measured using anautomatic analyzer (7170S; Hitachi Ltd.) in accordance with thefollowing procedure.

To 7.5 μL of each sample, 150 μL of reagent 1 for measuring an LDactivity [50 mmol/L phosphate buffer (pH 7.50) and 0.25 mmol/L NADH] wasadded and incubated at 37° C. for 5 minutes, and then 30 μL of reagent 2for measuring an LD activity [50 mmol/L phosphate buffer (pH 7.50) and12 mmol/L lithium salt of pyruvic acid] was further added, stirred, andincubated at 37° C. for 5 minutes. After approximately 1 minute to 2minutes from the addition of the reagent 2 for measuring an LD activity,an amount of change in absorbance per minute at the wavelength of 340 nmwas measured. Each residual ratio of the LD activity was calculated whenthe amount of change in absorbance at the first day of incubation wasregarded as 100%.

The results are shown in FIG. 3. In FIG. 3, the terms “1W”, “2W”, and“3W” mean the results after 1 week, 2 weeks, and 3 weeks from thebeginning of the incubation, respectively.

As shown in FIG. 3, the residual ratio of the LD activity in the reagentB of the present invention was higher than that in the reagent A forcomparison, both at 37° C. and at 25° C.

INDUSTRIAL APPLICABILITY

According to the reagent for measuring an ALT activity of the presentinvention, an increased reagent blank reaction, i.e., an increasedinitial absorbance, can be suppressed, and thus accurate measured valuesof the ALT activity can be obtained. Further, the reagent of the presentinvention exhibits an activity of stabilizing LD.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

1. In a method for measuring an alanine aminotransferase activity in asample, said method comprising the steps of: bringing the sample intocontact with L-alanine, 2-oxoglutaric acid, lactate dehydrogenase, andreduced nicotinamide adenine dinucleotide, generating pyruvic acid andL-glutamic acid from L-alanine and 2-oxoglutaric acid as substrates byalanine aminotransferase, converting pyruvic acid and reducednicotinamide adenine dinucleotide to lactic acid and oxidizednicotinamide adenine dinucleotide by lactate dehydrogenase, andmeasuring a decreased amount of reduced nicotinamide adeninedinucleotide or an increased amount of oxidized nicotinamide adeninedinucleotide, the improvement comprising bringing the sample intocontact with L-alanine, 2-oxoglutaric acid, lactate dehydrogenase, andreduced nicotinamide adenine dinucleotide in the presence of a substancehaving an activity of inhibiting a lactate dehydrogenase activity, saidsubstance being selected from the group consisting of oxamic acid,oxalic acid, oxalacetic acid, pyruvic acid, phosphoenolpyruvic acid,sodium dodecyl sulfate, lactic acid, and hydroxyglutaric acid, and saltsthereof, and the concentration of said substance in the measuring systembeing a concentration exhibiting the lactate dehydrogenase activitywhich does not affect the measurement of the alanine aminotransferaseactivity.
 2. The method according to claim 1, wherein the substancehaving an activity of inhibiting a lactate dehydrogenase activity isoxamic acid or a salt thereof.
 3. The method according to claim 2,wherein a concentration of oxamic acid or a salt thereof is 0.005 to 5mmol/L as a final concentration in the measuring system.
 4. The methodfor measuring an alanine aminotransferase activity according to claim 1,wherein a concentration of lactate dehydrogenase is 100 U/L or more as afinal concentration in the measuring system.